应用b-FGF刺激的软骨细胞构建自体组织工程化软骨的研究

Regeneration of autologous tissue-engineered cartilage by using basic-fibroblast growth factor in vitro culture

目的 探索碱性成纤维细胞生长因子 (b FGF)对体外软骨细胞增殖和体内组织工程化软骨构建的影响。 方法 细胞取自猪耳软骨 ,用Ham′sF 12培养液体外培养 ,实验分两组 :培养液含b FGF组 (b FGF组 ) ,培养液无b FGF组 (对照组 )。培养过程中观察细胞形态 ,于 (12 75± 1 2 6 )d后分别收集 2组第 2代细胞 ,进行细胞增殖倍数比较 ,并按 5 0× 10 6 cell ml浓度与聚环氧乙烯 (Plu ronic)混合 ,制成细胞 生物材料复合物 ,按每一样本 0 5ml注射到猪自体皮下 ,8周后取材 ,从重量、体积、GAG含量和组织学等方面进行分析。结果 b FGF组细胞贴壁呈类成纤维细胞样 ,对照组细胞贴壁呈多角形 ;各组第 2代细胞数和原代细胞数相比 ,b FGF组扩增 70倍 ,对照组为 5 4倍 ,b FGF组是对照组的 12 7倍 ;8周后取材 ,b FGF组形成的组织工程化软骨的平均重量和体积为 0 371g 0 370cm3,约是对照组的 2倍 ;GAG含量和组织学上 ,两者均非常接近正常猪耳软骨。结论 培养液中应用b FGF ,原代软骨细胞能在 2周内大量扩增 ,并能在体内构建良好的软骨 ,这有助于解决软骨组织工程种子细胞缺乏问题。

Objective To investigate the effect of the basic fibroblast growth factor (b-FGF ) to regenerate an autologous tissue-engineered cartilage in vitro. Methods The Cells were harvested from the elastic auricular cartilage of swine,and were plated at the concentration of 1x10 4 cells/cm 2,studied in vitro at two different media enviroments : Group I contained Ham ′s F-12 with supplements and b-FGF,Group II contained Ham′s F-12 only with supplements. The passage 2 cells (after 12.75±1.26 days )were harvested and mixed with 30% pluronic F-127/Ham′s F-12 at the concentration of 50×10 6 cells/ml. It was injected subcutaneously at 0.5 ml per implant. The implants were harvested 8 weeks after the vivo culture and examined with the histological stains. Results The chondrocytes displayed morphologically similar to the fibroblasts in the media containing basic-FGF. The number of cell doublings (after 12.75±1.26 days )in vitro culture was as the following: Group I,70;Group II,5.4. Eight 8 weeks after the vivo autologous implantation,the average weight (g) and volume (cm 3 ) in each group was as the following: Group I,0.371?g/0.370 cm 3 Group II,0.179 g/0.173 cm 3( P <0.01). With the b-FGF in vitro culture,the cells were expanded by 70 times after 2 weeks. Histologically,all of the engineered cartilage in the two groups were similar to the native elastic cartilage. Conclusion These results indicate that the basic-FGF could be used positively to enhance the quality and quantity of the seeding cells for the generation of the well-engineered cartilage. ;